The cell cycle ensures faithful duplication and segregation of duplicated chromosomes into daughter cells. Dysregulation of the cell cycle results in genomic instability, including aneuploidy, a hallmark of cancer and a significant cause of infertility and genetic syndromes when aneuploidy arises in the early embryo or gametes. ES cells maintain genomic stability even though they have altered Rb and p53 function and they are able to escape senescence, dividing indefinitely without the accruing DNA damage. This research proposal will test the hypothesis that hES cells possess unique and specialized mechanisms to maintain genomic stability. My career goal is to become an independently funded clinician-scientist in the field of stem cell biology with expertise in hES and induced pluripotent stem (iPS) cell manipulation and cell cycle regulation. The pathway to Independence Award (K99/R00) will greatly assist my transition to full independence. In the immediate future, this award will allow me to gain in-depth expertise in pluripotent cell biology, live-cell imaging and the advanced molecular biological techniques to study cell cycle regulation under the mentorship of Dr. George Q. Daley and co-mentorship of Dr. Marc W. Kirschner. The training program in the hematology/oncology division at Children's Hospital and department of systems biology at Harvard Medical School brings together resources from both institutions as well as those ofthe Harvard Stem Cell Institute, and provides an outstanding environment for the completion of training during the mentored phase. This will greatly facilitate my effective transition to independence. ^ The specific aims of this research proposal are to: 1) characterize the Rb and p53 pathways and their role in genomic stability maintenance in human pluripotent cells, and 2) generate IPS cells from somatic cells that harbor mutations implicated in genomic instability. Advanced techniques, including live and fixed cell imaging, will be applied to human pluripotent stem cells to perform in depth analysis ofthe pathways maintaining genomic stability. Viral-mediated gene over expression / RNAi gene knock-down techniques and small molecules will be used to interrogate these pathways and will be complemented by the development genomic instability disease specific iPS cell lines. The goal of this research is to study the mechanisms of genomic stability maintenance in pluripotent cells in order to better understand the molecular basis of aneuploidy.